Glycogen synthase kinase-3 inhibitors

Abstract

Novel peptide inhibitors of GSK-3, compositions containing same and uses thereof are disclosed. The novel peptide inhibitors are converted to inhibitors of GSK-3 upon interacting with the enzyme's catalytic site and hence act as disease-selective inhibitors for treating conditions associated with increased activity and/or expression of GSK-3. Each of the disclosed peptides is independently of no more than 15 amino acid residues, and has an amino acid sequence which comprises a ZX 1 X 2 X 3 Z(p) recognition motif of GSK-3, wherein Z(p) is a phosphorylated serine or threonine residue; Z is a phosphorylatable serine or threonine residue, and each of X 1 , X 2 and X 3 is independently any amino acid, as defined in the specification. Further disclosed are methods of identifying a putative substrate-competitive peptide inhibitor of GSK-3 which are effected by computational modeling and screening.

What is claimed is: 
     
       1. A peptide having the amino acid sequence II:
   [Y n   ••• Y 3 Y 2 Y 1 ]ZX 1 X 2 X 3 Z(p)[W 1   ••• W m]   (II)
 
 wherein: 
 n is 5, 6 or 7, 
 m is 1, 2, 3, 4 or 5; 
 m and n are such that the peptide consists of no more than 15 amino acid residues; 
 Z(p) is a phosphorylated serine residue or a phosphorylated threonine residue; 
 Z is a serine residue or a threonine residue; 
 X 1 , X 2 , X 3  and W 1 -Wm are each independently any amino acid residue; and 
 Y 1 -Yn comprises an amino acid sequence as set forth in SEQ ID NO:8: 
 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 8) 
                 
                     
                   Lys-Glu- Y   3 -Pro-Pro 
                 
             
                
                
               
            
           
         
         wherein Y 3  is any amino acid residue excepting a glutamic acid residue. 
       
     
     
       2. The peptide of  claim 1 , wherein Y 3  is an amino acid residue having a Log P higher than −3. 
     
     
       3. The peptide of  claim 1 , wherein Y 3  is an alanine residue. 
     
     
       4. The peptide of  claim 1 , wherein at least one of X 1 , X 2 , X 3  and W 1 -Wm is identical to a corresponding amino acid sequence of HSF-1. 
     
     
       5. The peptide of  claim 1 , wherein Z is a serine residue. 
     
     
       6. The peptide of  claim 1 , wherein Z(p) is a phosphorylated serine residue. 
     
     
       7. The peptide of  claim 1 , having an amino acid sequence as set forth in SEQ ID NO:9. 
     
     
       8. The peptide of  claim 1 , further comprising a hydrophobic moiety attached thereto. 
     
     
       9. The peptide of  claim 8 , having the amino acid sequence as set forth in SEQ ID NO:10. 
     
     
       10. A pharmaceutical composition comprising, as an active ingredient, the peptide of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
       11. A pharmaceutical composition comprising, as an active ingredient, a peptide, being of no more than 15 amino acid residues, and having an amino acid sequence which comprises a ZX 1 X 2 X 3 Z(p) recognition motif of a native GSK-3 substrate, wherein Z(p) is a phosphorylated serine residue or a phosphorylated threonine residue; Z is a serine residue or a threonine residue, and each of X 1 , X 2  and X 3  is any amino acid,
 the peptide having an amino acid sequence upstream said Z in which at least one polar or charged amino acid residue in an amino acid sequence upstream said serine or threonine residue of said native GSK-3 substrate is replaced by at least one amino acid residue selected from the group consisting of a non-polar amino acid residue, a non-charged amino acid residue (at a physiological pH) and a hydrophobic amino acid residue, 
 the peptide exhibiting an inhibition activity towards GSK-3, such that when a GSK-3 reacts with a GSK-3 substrate in the presence of the peptide, an apparent Michaelis Constant (app Km value) of the GSK-3 substrate is higher than a Michaelis Constant (Km value) obtained in a similar assay in the absence of the peptide inhibitor, 
 and a pharmaceutically acceptable carrier. 
 
     
     
       12. A method of inhibiting an activity of GSK-3, the method comprising contacting cells expressing GSK-3 with an effective amount of the peptide of  claim 1 . 
     
     
       13. A method of inhibiting an activity of GSK-3, the method comprising contacting cells expressing GSK-3 with an effective amount of a peptide, being of no more than 15 amino acid residues, and having an amino acid sequence which comprises a ZX 1 X 2 X 3 Z(p) recognition motif of a native GSK-3 substrate, wherein Z(p) is a phosphorylated serine residue or a phosphorylated threonine residue; Z is a serine residue or a threonine residue, and each of X 1 , X 2  and X 3  is any amino acid,
 the peptide having an amino acid sequence upstream said Z in which at least one polar or charged amino acid residue in an amino acid sequence upstream said serine or threonine residue of said native GSK-3 substrate is replaced by at least one amino acid residue selected from the group consisting of a non-polar amino acid residue, a non-charged amino acid residue (at a physiological pH) and a hydrophobic amino acid residue, 
 the peptide exhibiting an inhibition activity towards GSK-3, such that when a GSK-3 reacts with a GSK-3 substrate in the presence of the peptide, an apparent Michaelis Constant (app Km value) of the GSK-3 substrate is higher than a Michaelis Constant (Km value) obtained in a similar assay in the absence of the peptide inhibitor. 
 
     
     
       14. A method of treating a biological condition associated with GSK-3 activity, the method comprising administering to a subject in need thereof a therapeutically effective amount of the peptide of  claim 1 . 
     
     
       15. A method of treating a biological condition associated with GSK-3 activity, the method comprising administering to a subject in need thereof a therapeutically effective amount of a peptide, being of no more than 15 amino acid residues, and having an amino acid sequence which comprises a ZX 1 X 2 X 3 Z(p) recognition motif of a native GSK-3 substrate, wherein Z(p) is a phosphorylated serine residue or a phosphorylated threonine residue; Z is a serine residue or a threonine residue, and each of X 1 , X 2  and X 3  is any amino acid,
 the peptide having an amino acid sequence upstream said Z in which at least one polar or charged amino acid residue in an amino acid sequence upstream said serine or threonine residue of said native GSK-3 substrate is replaced by at least one amino acid residue selected from the group consisting of a non-polar amino acid residue, a non-charged amino acid residue (at a physiological pH) and a hydrophobic amino acid residue, 
 the peptide exhibiting an inhibition activity towards GSK-3, such that when a GSK-3 reacts with a GSK-3 substrate in the presence of the peptide, an apparent Michaelis Constant (app Km value) of the GSK-3 substrate is higher than a Michaelis Constant (Km value) obtained in a similar assay in the absence of the peptide inhibitor. 
 
     
     
       16. A peptide having the amino acid sequence II*:
   [Y n   ••• Y 3 Y 2 Y 1 ]Z(p) 1 X 2 X 3 Z(p) 2 [W 1   ••• W m]   (II*)
 
 wherein: 
 n is 5, 6 or 7, 
 m is 1, 2, 3, 4 or 5; 
 m and n are such that the peptide consists of no more than 15 amino acid residues; 
 Z(p) 1  and Z(p) 2  are each independently a phosphorylated serine residue or a phosphorylated threonine residue; 
 X 1 , X 2 , X 3  and W 1 -Wm are each independently any amino acid residue; and 
 Y 1 -Yn comprises an amino acid sequence as set forth in SEQ ID NO:8: 
 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 8) 
                 
                     
                   Lys-Glu- Y   3 -Pro-Pro 
                 
             
                
                
               
            
           
         
         wherein Y 3  is any amino acid residue excepting a glutamic acid residue. 
       
     
     
       17. The composition of  claim 11 , wherein said at least one amino acid sequence upstream said Z is such that when said Z is phosphorylated upon interaction with the catalytic binding site of GSK-3, a phosphorylated peptide obtained by said interaction exhibits a dissociation constant lower than a dissociation constant of said native GSK-3 substrate by at least 2-folds. 
     
     
       18. The composition of  claim 11 , wherein said amino acid residue upstream said Z is selected from the group consisting of alanine residue, glycine residue, valine residue, leucine residue, isoleucine residue, proline residue, methionine residue, cysteine residue, tryptophan residue, tyrosine residue and phenylalanine residue. 
     
     
       19. The composition of  claim 11 , having an amino acid sequence I:
   [Y n   ••• Y 3 Y 2 Y 1 ]ZX 1 X 2 X 3 Z(p)[W 1   ••• W m]   (I)
 
 wherein: 
 m equals 1, 2, 3, 4 or 5; 
 n is 3, 4, 5, 6 or 7, such that m and n are such that said peptide consists of no more than 15 amino acid residues; 
 Z(p) is said phosphorylated serine residue or said phosphorylated threonine residue; 
 Z is said serine residue or said threonine residue; 
 X 1 , X 2 , X 3 , Y 1 -Yn and W 1 -Wm are each independently an amino acid residue of said substrate, except that at least one charged or polar amino acid residue of said Y 1 -Yn in said native GSK-3 substrate is replaced by said at least one amino acid residue selected from the group consisting of a non-polar amino acid residue, a non-charged amino acid residue (at a physiological pH) and a hydrophobic amino acid residue. 
 
     
     
       20. The composition of  claim 11 , wherein said peptide further comprises a hydrophobic moiety attached thereto. 
     
     
       21. The method of  claim 13 , wherein said at least one amino acid sequence upstream said Z is such that when said Z is phosphorylated upon interaction with the catalytic binding site of GSK-3, a phosphorylated peptide obtained by said interaction exhibits a dissociation constant lower than a dissociation constant of said native GSK-3 substrate by at least 2-folds. 
     
     
       22. The method of  claim 13 , wherein said amino acid residue upstream said Z is selected from the group consisting of alanine residue, glycine residue, valine residue, leucine residue, isoleucine residue, proline residue, methionine residue, cysteine residue, tryptophan residue, tyrosine residue and phenylalanine residue. 
     
     
       23. The method of  claim 13 , having an amino acid sequence I:
   [Y n   ••• Y 3 Y 2 Y 1 ]ZX 1 X 2 X 3 Z(p)[W 1   ••• W m]   (I)
 
 wherein: 
 m equals 1, 2, 3, 4 or 5; 
 n is 3, 4, 5, 6 or 7, such that m and n are such that said peptide consists of no more than 15 amino acid residues; 
 Z(p) is said phosphorylated serine residue or said phosphorylated threonine residue; 
 Z is said serine residue or said threonine residue; 
 X 1 , X 2 , X 3 , Y 1 -Yn and W 1 -Wm are each independently an amino acid residue of said substrate, except that at least one charged or polar amino acid residue of said Y 1 -Yn in said native GSK-3 substrate is replaced by said at least one amino acid residue selected from the group consisting of a non-polar amino acid residue, a non-charged amino acid residue (at a physiological pH) and a hydrophobic amino acid residue. 
 
     
     
       24. The method of  claim 13 , wherein said peptide further comprises a hydrophobic moiety attached thereto. 
     
     
       25. The method of  claim 15 , wherein said at least one amino acid sequence upstream said Z is such that when said Z is phosphorylated upon interaction with the catalytic binding site of GSK-3, a phosphorylated peptide obtained by said interaction exhibits a dissociation constant lower than a dissociation constant of said native GSK-3 substrate by at least 2-folds. 
     
     
       26. The method of  claim 15 , wherein said amino acid residue upstream said Z is selected from the group consisting of alanine residue, glycine residue, valine residue, leucine residue, isoleucine residue, proline residue, methionine residue, cysteine residue, tryptophan residue, tyrosine residue and phenylalanine residue. 
     
     
       27. The method of  claim 15 , having an amino acid sequence I:
   [Y n   ••• Y 3 Y 2 Y 1 ]ZX 1 X 2 X 3 Z(p)[W 1   ••• W m]   (I)
 
 wherein: 
 m equals 1, 2, 3, 4 or 5; 
 n is 3, 4, 5, 6 or 7, such that m and n are such that said peptide consists of no more than 15 amino acid residues; 
 Z(p) is said phosphorylated serine residue or said phosphorylated threonine residue; 
 Z is said serine residue or said threonine residue; 
 X 1 , X 2 , X 3 , Y 1 -Yn and W 1 -Wm are each independently an amino acid residue of said substrate, except that at least one charged or polar amino acid residue of said Y 1 -Yn in said native GSK-3 substrate is replaced by said at least one amino acid residue selected from the group consisting of a non-polar amino acid residue, a non-charged amino acid residue (at a physiological pH) and a hydrophobic amino acid residue. 
 
     
     
       28. The method of  claim 15 , wherein said peptide further comprises a hydrophobic moiety attached thereto.